Abstract The development and viability of multicellular organisms requires robust, accurate control of gene expression. Although some of the information governing this process is encoded within the DNA, much is passed on `epigenetically' (i.e. independent of the DNA sequence). Importantly, histone post-translational modifications (PTMs) modulate the organization of chromatin and are widely hypothesized to be essential carriers of epigenetic information. Until recently, it has been impossible to rigorously test this premise in multicellular eukaryotes, because the repetitive nature of histone gene clusters makes them difficult to manipulate genetically. However, we have developed an innovative genetic platform in Drosophila melanogaster that allows direct interrogation of the function of specific histone residues. We can now study the biological function of a specific histone PTM, by changing the acceptor residue to an amino acid that cannot be appropriately modified. Critically, our approach enables all wild-type copies of that histone gene to be replaced with mutant copies. In this proposal, we investigate the function of histone H3K36 methylation (H3K36me) in maintaining metazoan transcriptome fidelity. Specific aims for the proposal leverage our novel platform to directly assay the roles of both replication- dependent H3K36 and -independent H3.3K36 histone PTMs in transcription, pre-mRNA processing and mRNA stability. This research is important for human health because mutations in H3K36 and the evolutionarily conserved enzymes that catalyze modification of this residue (e.g. Set2 and NSD) are implicated in many human diseases, including cancer.